Two factors have contributed to the urgent need for effective new antifungal agents - the rising incidence of systemic yeast and mold infections, especially in immunocompromised individuals, and the current lack of drugs that are both fungicidal and relatively free of deleterious side effects. The aim of the proposed research is to identify novel small molecule inhibitors of fungal RNA polymerase II, the enzyme required for the synthesis of all nuclear-encoded mRNAs. The rationale for targeting RNA polymerase II is based on its requirement for fungal viability, genetic and biochemical evidence demonstrating mechanistic differences between fungal and mammalian RNA polymerase II transcription and the apparent lack of prior research into antifungal therapeutics targeted specifically against RNA polymerase II. The three major steps toward realizing the aim of this proposal are: l) purification of RNA polymerase II from pathogenic fungi in sufficient quantity to support high-throughput screening; 2) development of a robust, high-throughput biochemical screen for compounds that selectively inhibit fungal, but not human, RNA polymerase II; 3) adaptation of the assay to suit the requirements of defined chemical and natural product extract library screening. PROPOSED COMMERCIAL APPLICATION: Systemic fungal infections are a major cause of pathology and mortality in immunocompromised individuals. The available antifungal agents target a very limited number of metabolic pathways and have one or more major limitations, including fungistatic - rather than fungicidal - activity, high toxicity, development of resistance and narrow spectrum of activity. The intent of the proposed research is to develop a high-throughput screen for novel therapeutics directed against RNA polymerase II of fungal pathogens.